CA2163966A1 - Alkaline and acid phosphatase inhibitors in treatment of neurological disorders - Google Patents
Alkaline and acid phosphatase inhibitors in treatment of neurological disordersInfo
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- CA2163966A1 CA2163966A1 CA002163966A CA2163966A CA2163966A1 CA 2163966 A1 CA2163966 A1 CA 2163966A1 CA 002163966 A CA002163966 A CA 002163966A CA 2163966 A CA2163966 A CA 2163966A CA 2163966 A1 CA2163966 A1 CA 2163966A1
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Abstract
The present invention provides a method of inhibiting .beta.-amyloid toxicity in brain cells. The method includes administering to the cells an amount of an alkaline phosphatase inhibitor which is pharmacologically effective to reduce degeneration in the cells. Methods of treatment of peripheral neuropathy are also provided using acid or alkaline phosphatase inhibitors.
Description
WO 94/27603 ' ~ 2 1 6 3 9 6 ~ PCTJUS94/06186 ALKALINE AND ACID PHOSPHATASE INHIBITORS
IN TREATMENT OF NEUROLOGICAL DISORDERS
Field of the Invention The present invention relates to the use of alkaline and acid phosphatase inhibitors 5 in the treatment of neurological disorders. More particularly, the present invention relates to the use of various specific phosphatase inhibitors in the treatment of ~-amyloid toxicity in brain cells and the use of phosphatase inhibitors in the treatment of peripheral neuropathy.
Backaround of the Invention Alzheimer's Disease (AD) is a progressive neurodegenerative condition affecting a substantial proportion of people over the age of 65. There is presently no known cure.
One hallmark of AD neuropathology includes neurofibrillary tangles involving neuronal processes and closely associated with gliotic astrocytes and with macrophages. Another hallmark of AD is the presence in the brain of affected individuals of "plaques" composed of a variety of proteins, glycoproteins, and other components. These plaques always contain a high proportion of,~-amyloid peptide, a 42-43 amino acid peptide aberrantly cleaved from the amyloid precursor protein.
Though normally observed in blood and cerebrospinal fluid, ~-amyloid peptide in an aggregated form is considered partly responsible for the noted neuronal deathobserved in AD (Koh, et al., Brain Res. 533:315 (1990); Mattson, et al., J. Neurosoi, 12:376 1992, etc.). Exposure of brain cells cultured in vitro to ~-amyloid peptide results in the degeneration of these cells. How this aggregated peptide mediates neuronal death is unclear, yet published reports suggest a programmed cell death or apoptotic mechanism. Loo, et al., PNAS, 90:7951 (1993).
Neuronal death found in AD involves the loss of specific neuronal subtypes, possibly by the aforementioned mechanism of programmed cell death/apoptosis (PCD), an active suicide program that differs markedly from necrotic death (Loo, et al., PNAS
90:7951, 1993). The mechanism of programmed cell death induction in neurons is unclear, yet measurable/observable characteristics of PCD exist. Examples of changes observed during the process of PCD are chromatin condensation or margination, synthesis of required death proteins, DNA fragmentation into repeating 200 bp units. Increases in certain transcription and translation products that precede the programmed death are also obser~/ed.
Other amyloidogenic diseases exist that are associated with ,t~-amyloid. These include Downs Syndrome and Hereditary Cerebral Hemorrhage with Amyloidosis-DutchType (HCHWA-D).
WO 94/27603 ~ 6 3 q 6 6 PCT/US94/06186 Alkaline phosphatases are a family of enzymes which serve diverse functions in mammalian cells. The major activity of these enzymes is to remove phosphate fromnucleotides, and the 5' nucleotidases are included in this family. While specific chemical reactions catalyzed by these enzymes are well characterized, their role in cellular metabolism and regulation is less clearly defined.
Levamisole is an alkaline phosphatase inhibitor sold commercially under the brand name "Ergamisol" by Janssen Pharmaceutica of Piscataway, New Jersey. This product is available in tablets of levamisole hydrochloride for oral administration that contain the equivalent of 50 mg of levamisole base. Levamisole has been used in the treatment of colon cancer. Recommended dosage for treatment of colon cancer is 50 mg every eight hours for three days.
Acid phosphatases are another family of enzymes that serve diverse functions in mammalian cells.
European patent Application No. 91107844.2, Publication No. 0457295A2, purports to disclose the use of protein phosphatase inhibitors, such as okadaic acid, calyculin-A and vanadate for the treatment of amyloidosis associated with AD.
Summarv of the Invention The present invention relates to the finding that arylimidothiazole derivative inhibitors of alkaline phosphatase can reduce neuronal death induced by ,~-amyloid peptide.
In a first aspect, the present invention is a method of inhibiting ,6-amyloid toxicity in brain cells. This method includes administering to the cells an amount of an arylimidothiazole derivative having alkaline phosphatase inhibitory activity effective to reduce cell degeneration. The brain cells can be located in a living organism suffering from an amyloidogenic neurologic disorder, such as Alzheimer's Disease (AD). The brain cells can also be located in a living organism suffering from other amyloidogenic diseases, such as Downs Syndrome or Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch Type (HCHWA-D). The brain cells can also be located in vitro. In a preferred embodiment, the arylimidothiazole derivative is administered orally, intravenously, intraperitoneally, intramuscularly, by injection into cerebrospinal fluid, by injection into an intracerebral ventricle, or via a carrier protein. The arylimidothiazole derivative is preferably administered at a dose of from approximately 0.01 mg/kg to 10 mg/kg body weight per day, more preferably less than 5 mg/kg. Preferred arylimidothiazole derivatives include levamisole, bromolevamisole, and bromotetramisole.
In a second aspect, the invention is a method of treating a human subject havinga peripheral neuropathy. This method includes administering to the subject an amount ~ ~ r~- -2 ~ 6 3 9 6 6 PCT/US94/06186 of an alkaline phosphatase inhibitor which is pharmacologically effective to alleviate the symptoms of the peripheral neuropathy. The alkaline phosphatase inhibitor can beadministered in any of a variety of ways, including orally, intravenously, intraperitoneally, or intramuscularly. Preferred alkaline phosphatase inhibitors for this aspect of the 5 invention are arylimidothiazole derivatives, such as levamisole, bromolevamisole and bromotetramisole. The alkaline phosphatase inhibitor is preferably administered at a dose of from approximately 0.001 mg/kg to 10 mg/kg body weight per day, more preferably less than 5 mg/kg.
In a third aspect the invention is a method of treating a human subject having aperipheral neuropathy. This method includes administering to the subject an amount of an acid phosphatase inhibitor which is pharmacologically effective to alleviate the symptoms of the peripheral neuropathy. The acid phosphatase inhibitor can be administered in any of a variety of ways, including orally, intravenously, intraperitoneally, or intramuscularly. Preferred acid phosphatase inhibitors for this aspect of the invention 1 5 are para-chloromercuribenzoate (pCMB) and inorganic salts of tal l, ale or fluoride.
Preferred inorganic salts for this aspect of the invention are Na; K; and Na,K. The acid phosphatase inhibitor is preferably administered at a dose of from approximately 0.001 mg/kg to 10 mg/kg body weight per day.
Brief DescriPtion of the Drawinqs FIGURE 1 graphically depicts the effect of alkaline phosphatase inhibitors on rat brain neurotumor cells treated with ~-amyloid peptide.
FIGURE 2 graphically depicts the effect of levamisole on primary cultures of ratembryonic hippocampal neurons exposed to ~-amyloid peptide.
FIGURE 3 graphically depicts the effect of okadaic acid on hippocampal neurons treated with,~-amyloid peptide.
FIGURE 4 graphically depicts the effect of levamisole or vanadate on alkaline phosphatase activity from porcine kidney and from calf intestine.
FIGURE 5 graphically depicts the effect of Na,K-ta~l~ate on dorsal root ganglioncells treated with Nerve Growth Factor and vinblastine.
Detailed Description of the Invention The present invention relates to the use of alkaline phosphatase inhibitors or acid phosphatase inhibitors as therapeutic agents for neurological disorders, such as Alzheimer's Disease (AD) and peripheral neuropathies.
It is one of the surprising discoveries of the present invention that arylimidothiazole derivatives having alkaline phosphatase inhibitory activity can be used to inhibit the death of neurons due to,~-amyloid peptide toxicity.
~ 639 66;
WO 94/27603 . = . PCTIUS94/06186 A number of different approaches can be taken to the inhibition of alkaline phosphatases. For example, they can be inhibited by arylimidothiazole compounds such as levamisole, which is specific for mammalian alkaline phosphatase, or by compounds that complex active Zn ion, which is required for alkaline phosphatase activity. Chemical 5 inhibitors include, but are not limited to,~ ~vàmisole I (-)-(S)-2,3,5,6-tetrahydro-6-phenylimidoazide [2,1-b] thiazole -rnonohydrochloride), bromolevamisole, bromotet,a",isole, other arylimidothiazole derivatives, and the like. Still other alkaline phosphatase inhibitors include 1,10 phenanthroline, phenylalanine, IBMX, purines, and theophylline.
The presence of ,6-amyloid in AD and other amyloidogenic proteins is associated not only with deposition of amyloid in plaques, but also with neurotoxicity associated with this peptide. Thus, prevention of this neurotoxicity is an important goal of the present invention. We have surprisingly discovered that arylimidothiazole derivatives having alkaline phosphatase inhibitory activity can attenuate or prevent such 1 5 neurotoxicity.
Another aspect of the present invention involves administration of acid phosphatase inhibitors to patients with peripheral neuropathies.
A number of different approaches can be taken to the inhibition of acid phosphatases. Acid phosphatase can be inhibited by chemical compounds, such as para-chloromercuribenzoate (pCMB) or inorganic salts of tartrate and fluoride. Preferred inorganic salts include Na; K; and Na,K.
One aspect of the present invention involves administration of inhibitors of alkaline phosphatase to patients with an amyloidogenic disease associated with ,B-protein, including AD, Downs Syndrome and HCHWA-D, as well as administration to patients suspected of having these diseases, particularly AD.
Alzheimer's Disease can be diagnosed by any number of traditional diagnostic criteria such as psychometrics that assess, for example, cognitive function, intelligence, language, memory, visual-spatial skills, and frontal lobe skills, or by neuropathological criteria such as immunological markers, enzyme assay, or CAT scan.
The invention can also be used prophylactically for those patients at risk for the development of AD or other amyloidogenic disease. Such patients can be identified through family histories or through a biochemical or immunological test indicative of such diseases. An example of an immunological test for AD involves the use of an antibody for the amyloid precursor protein (APP) in the CSF of a patient suspected of having AD.
In this test, low levels of APP compared to normal levels are indicative of AD. See Van Nostrand et al., Science, 256:1279 (1992).
WO 94/27603 2 1 6 3 9 6 ~ ~` PCT/US94/06186 The administration of alkaline phosphatase inhibitors to prevent~-amyloid peptide toxicity can be by any of a number of methods known to those skilled in the art. Such methods include the delivery of the inhibitor orally or through intravenous, intraperitoneal, or intramuscular injection. Other known methods include techniques for administering compounds directly into the central nervous system (CNS), such as injection intocerebrospinal fluid, injection into an intracerebral ventricle, or by a carrier protein that actively transports the inhibitor to the brain. Admihistration of alkaline or acid phosphatase inhibitors for treatment of peripheral neuropathies can be by any of the foregoing; however, such treatment generally does not involve admir~ ion into the 1 0 CNS.
P~efer,ed dosage range when using chemical inhibitors can be determined using techniques known to those having ordinary skill in the art. For levamisole, dosages of 0.001 mg/kg to 10 mg/kg body weight per day for an average adult, more preferably less than 5 mg/kg, are believed to be effective in the treatment of AD. For some patients daily doses of an alkaline phosphatase inhibitor is required for optimum relief. For other patients relief can be achieved with every other day, or even weekly administration.
It is known that cultured rat hippocampal neurons are killed by the application of ~-amyloid peptides. Thus, prevention of neurotoxicity can be shown as an inhibition in death of these neurons. Selection of appropriate alkaline phosphatase inhibitors for use in inhibitors of ,~-amyloid toxicity can thus be made through use of a model system making use of hippocampal neurons in the presence of,~-amyloid peptides. Compounds that prevent death of these neurons in this system are likely to have significant activity in inhibition of ~-amyloid toxicity.
The effect of treatment with alkaline phosphatase inhibitors on neurons exposed to,t~-amyloid peptide was tested in two experiments, described below in Examples 1 and 2.
Examnle 1 Inhibition of ~-amYloid PePtide Toxicitv bY Alkaline Phosphatase Inhibitors HT4 neurotumor cells were treated with 50~uM,B-amyloid peptide 25-35 and 250 ~M quisqualate. The cells were also treated with 100 ~M of the following alkaline phosphatase inhibitors: 1,10 phenanthroline, phenylalanine, IBMX, bromotetramisole, purine, and theophylline. The death of the neurons was determined by measuring LDH
activity in the media 24 hours after treatment. The release of LDH from the cells into the surrounding media indicates cell death.
WO 94/27603 2 ~ 6 3 9 6 6 s PCTIUS94/06186 FIGURE 1 illustrates the results of the study of Example 1. Control cells, treated only with,~-amyloid peptide and quisqualate, showed a high LDH activity 24 hours after treatment, indicating a high degree of cell death. Cells subjected to no treatment had only a low level of LDH activity. The addition of alkaline phosphatase inhibitors 5 significantly reduced the amount of LDH act~vity and therefore inhibited the death of neuronal cells due to exposure to ,B-amyloid peptide. IBMX appeared to be the most effective compound tested in inhibiting cell death from amyloid toxicity.
ExamPle 2 Inhibition of B-amvloid PePtide Toxicitv bv Levamisole To further determine the effect of alkaline phosphatase inhibitors on ~-amyloid peptide toxicity, rat embryonic hippocampal neurons in culture were exposed to 50,uM
,B-amyloid 25-35 in the presence of levamisole concentrations of 250 ~M and 500,uM.
The results of the experiment of Example 2 are illustrated in FIGURE 2. The death 15of the neurons, as indicated by the release of LDH from the cells, was reduced by the presence of levamisole at a concentration of 250,uM. LDH activity was also reduced by the presence of levamisole at a concentration of 500,uM.
Example 2 demonstrates that death of neuronal cells due to exposure to,B-amyloidpeptide is reduced by the presence of levamisole.
20As mentioned earlier, the prior art indicates that okadaic acid may be useful in the treatment of amyloidosis associated with Alzheimer's Disease. The experiment of Example 3 below, however, illustrates that okadaic acid causes hippocampal neuronal injury upon administration with ,B-amyloid.
25ExamPle 3 Okadaic Acid Causes HiPPocamPal Neuronal Iniurv UPon Administration with ~-Amvloid Hippocampal neurons were treated with 50,uM ,B-amyloid (,~25-35) and 50 nM
okadaic acid for 24 hours in Dulbeccois Modified Eagle's (DME) medium at 37C.
30Following the 24-hour treatment period, an aliquot of media was removed for determination of LDH efflux from the neurons, which is an indicator of cell death. Each bar in FIGURE 3, except the bar representing okadaic acid alone, represents the mean of 4 wells ~t the standard deviation. The bar representing okadaic acid alone represents the mean of 2 wells ~ the standard deviation.
35FIGURE 3 illustrates that okadaic acid causes hippocampal neuronal injury uponadministration with ~-amyloid. This finding is particularly surprising in light of the prior WO 94/27603 ~ t 6 3 9 6 6 = PCT/US94/06186 art, wherein okadaic acid is indicated as being useful for the treatment of amyloidosis associated with Alzheimer's Disease.
The effect of different alkaline phosphatase inhibitors are compared below in Example 4.
Example 4 Inhibition of Porcine Kidnev and Calf Intestinal Alkaline PhosPhatase Porcine kidney (0.01 U) and calf intestinal (0.002 U) alkaline phosphatases wereassayed in the presence of levamisole (200,uM) or vanadate salt (5-500,uM) using pNPP
(5 mM) as sub~l~ate. The assay was carried out in pH 5.5 Iysis buffer. Each bar in FIGURE 4 represents the mean of quadruplicates i the standard deviation.
The results of the experiment of Example 4 are illustrated in FIGURE 4. Both levamisole and vanadate appear to inhibit porcine kidney alkaline phosphatase activity 15 from porcine kidney and calf intestine. Inhibition of porcine kidney alkaline phosphatase activity is unexpectedly high in the presence of levamisole. Vanadate at 100,uM
significantly inhibited calf intestinal alkaline phosphatase, while levamisole at 200~M had little effect.
The effec~ of treatment on humans with AD using arylimidothiazole derivatives 20 having alkaline phosphatase inhibitory activity is described below in Examples 5-9.
Example 5 Oral Treatment of AD with Arvlimidothiazole Derivatives Human subjects diagnosed as suffering from AD are administered 50 mg of an 25 arylimidothiazole derivative, such as levamisole, orally every eight hours for three days.
The treatment is repeated every 14 days for a period of 6 months. With treatment, the symptoms of AD in the subject are lessened, as the subject experiences improved mernory and cognitive skills.
ExamPle 6 Intravenous Treatment of AD
with ArYlimidothiazole Derivatives Human subjects diagnosed as suffering from AD are administered 0.5 mg/kg arylimidothiazole derivatives intravenously once or twice daily for four days. This administration is repeated every 14 days for a period of 4 months.
- . ~
WO 94/27603 2 1 6 3 9 ~ ~ -8- PCT/US94/06186 Example 7 Treatment of AD with Levamisole A human patient diagnosed as suffering from Alzheimer's Disease is administered about 50 mg levamisole orally every eight hours for three days. The treatment is5 repeated every 14 days for a period of 6 months. The symptoms of AD in the patient are lessened, as the patient experiences improved memory and cognitive skills.
ExamPle 8 Continuous Treatment of AD
with Arylimidothiazole Derivatives Human subjects diagnosed as suffering from AD are administered 0.05 mg/kg of an arylimidothiazole derivative, such as levamisole, orally every day for a period of six months. With treatment, the symptoms of AD in the patient decrease and deterioration stops shortly after initiation of treatment.
ExamPle 9 Continuous Treatment of AD with Levamisole A human patient diagnosed as suffering from Alzheimer's Disease is identified ashaving deteriorating symptoms of the disease. The patient is administered about 10 mg 20 levamisole orally once per day for a period of six months. With treatment, the symptoms of AD in the patient decrease and deterioration stops shortly after initiation of treatment.
As discussed above, the present invention includes the treatment of peripheral neuropathies, such as chemotherapeutic induced neuropathy, and neurophathies caused by diabetes and other degenerative disease states. In the context of this aspect of the 25 invention, both acid and alkaline phosphatase inhibitors are effective. Exemplary inhibitors in this regard include Na, K-tartrate, NaF, calyculin-A, vanadate and the arylimidothiazole derivatives. Identification of appropriate protein phosphatase inhibitors can be conducted through a model system making use of dorsal root ganglion cellstreated with Nerve Growth Factor (NGF) and vinblastine. Effective compounds in this 30 system result in an increased percentage of cell viability in the presence of vinblastine.
The effect of treatment with Na,K-tartrate on dorsal root ganglion cells treatedwith Nerve Growth Factor and vinblastine was tested and is described below in Example 10.
WO 94/27603 2 t 6 3 9 6 6 ~ . ~ PCT/TJSg4/06186 g ExamPle 10 Inhibition of Vinblastine-lnduced Toxicity bv Na,K-Tartrate Dorsal root ganglion cells were treated with Nerve Growth Factor (NGF) (50 ng/ml), vinblastine (500 nM) and Na,K-tartrate (500 ,L~M) for 24 hours. Following the treal ment, the dorsal root ganglion cells were stained with ethidium bromide and fluorescein diacetate to stain dead and live cells, respectively.
FIGURE 5 illustrates the results of the study of Example 16. Percentage cell viability is determined from the ratio of live cells to total cell number. Each bar in FIGURE
7 represents the mean of 3 wells i the standard deviation. Controlcells,treatedonly with NGF, showed a high degree of cell viability. Cells exposed to NGF and vinblastine showed a decrease in cell viability. The addition of Na,K-tartrate significantly increased the percentage cell viability in the presence of vinblastine.
The effect of treatment on humans with peripheral neuropathy using either alkaline or acid phosphatase inhibitors is described below in Examples 1 1-14.
Example 1 1 Treatment of ChemotheraPeutic-lnduced PeriPheral Neuropathv with Acid Phosphatase Inhibitors Human subjects with peripheral neuropathy caused by chemotherapeutic agents such as vinblastine, vincristine, or taxol are administered 10 mg/kg of an acid phosphatase inhibitor. The acid phosphatase inhibitor is administered orally or systemically to these subjects once or twice a day for two days prior to and four days following the administration of the chemotherapeutic regimen. Following treatment, an assessment of motor coordination and peripheral paresthesia is made, as specified by those skilled in the art.
ExamPle 12 Treatment of NonchemotheraPeutic-lnduced PeriPheral NeuroPathv with Acid Phosphatase Inhibitors Human subjects with peripheral neuropathy caused by diabetes or other degenerative disease states are administered 5 mg/kg of an acid phosphatase inhibitor twice per day for a period of 6 months. Following treatment, an assessment of motor coordlination and peripheral paresthesia is made, as specified by those skilled in the art.
W094/27603 ~l 63966 PCT/US94/06186 ExamDIe 13 Treatment of ChemotheraPeutic-lnduced PeriPheral NeuroPathY with Alkaline Phosphatase Inhibitors Human subjects with peripheral neuropathy caused by chemotherapeutic agents 5 such as vinblastine, vincristine, or taxol are administered 5 mg/kg of levamisole. The alkaline phosphatase inhibitor is administered orally or systemically to these subjects once or twice a day for two days prior to and'~four days following the administration of the chemotherapeutic regimen. Following trèatment, an assessment of motor coordination and peripheral paresthesia is made, as specified by those skilled in the art.
Example 14 Treatment of Nonchemotherapeutic-lnduced PeriPheral NeuroPathY with Alkaline Phosphatase Inhibitors Human subjects with peripheral neuropathy caused by diabetes or other 15 degenerative disease states are administered 5 mg/kg of an alkaline phosphatase inhibitor. The alkaline phosphatase inhibitor is administered orally or systemically to the subjects once or twice a day for two days prior to and four days following the administration of the chemotherapeutic regimen. Following treatment, an assessment of motor coordination and peripheral paresthesia is made, as specified by those skilled in the 20 art.
IN TREATMENT OF NEUROLOGICAL DISORDERS
Field of the Invention The present invention relates to the use of alkaline and acid phosphatase inhibitors 5 in the treatment of neurological disorders. More particularly, the present invention relates to the use of various specific phosphatase inhibitors in the treatment of ~-amyloid toxicity in brain cells and the use of phosphatase inhibitors in the treatment of peripheral neuropathy.
Backaround of the Invention Alzheimer's Disease (AD) is a progressive neurodegenerative condition affecting a substantial proportion of people over the age of 65. There is presently no known cure.
One hallmark of AD neuropathology includes neurofibrillary tangles involving neuronal processes and closely associated with gliotic astrocytes and with macrophages. Another hallmark of AD is the presence in the brain of affected individuals of "plaques" composed of a variety of proteins, glycoproteins, and other components. These plaques always contain a high proportion of,~-amyloid peptide, a 42-43 amino acid peptide aberrantly cleaved from the amyloid precursor protein.
Though normally observed in blood and cerebrospinal fluid, ~-amyloid peptide in an aggregated form is considered partly responsible for the noted neuronal deathobserved in AD (Koh, et al., Brain Res. 533:315 (1990); Mattson, et al., J. Neurosoi, 12:376 1992, etc.). Exposure of brain cells cultured in vitro to ~-amyloid peptide results in the degeneration of these cells. How this aggregated peptide mediates neuronal death is unclear, yet published reports suggest a programmed cell death or apoptotic mechanism. Loo, et al., PNAS, 90:7951 (1993).
Neuronal death found in AD involves the loss of specific neuronal subtypes, possibly by the aforementioned mechanism of programmed cell death/apoptosis (PCD), an active suicide program that differs markedly from necrotic death (Loo, et al., PNAS
90:7951, 1993). The mechanism of programmed cell death induction in neurons is unclear, yet measurable/observable characteristics of PCD exist. Examples of changes observed during the process of PCD are chromatin condensation or margination, synthesis of required death proteins, DNA fragmentation into repeating 200 bp units. Increases in certain transcription and translation products that precede the programmed death are also obser~/ed.
Other amyloidogenic diseases exist that are associated with ,t~-amyloid. These include Downs Syndrome and Hereditary Cerebral Hemorrhage with Amyloidosis-DutchType (HCHWA-D).
WO 94/27603 ~ 6 3 q 6 6 PCT/US94/06186 Alkaline phosphatases are a family of enzymes which serve diverse functions in mammalian cells. The major activity of these enzymes is to remove phosphate fromnucleotides, and the 5' nucleotidases are included in this family. While specific chemical reactions catalyzed by these enzymes are well characterized, their role in cellular metabolism and regulation is less clearly defined.
Levamisole is an alkaline phosphatase inhibitor sold commercially under the brand name "Ergamisol" by Janssen Pharmaceutica of Piscataway, New Jersey. This product is available in tablets of levamisole hydrochloride for oral administration that contain the equivalent of 50 mg of levamisole base. Levamisole has been used in the treatment of colon cancer. Recommended dosage for treatment of colon cancer is 50 mg every eight hours for three days.
Acid phosphatases are another family of enzymes that serve diverse functions in mammalian cells.
European patent Application No. 91107844.2, Publication No. 0457295A2, purports to disclose the use of protein phosphatase inhibitors, such as okadaic acid, calyculin-A and vanadate for the treatment of amyloidosis associated with AD.
Summarv of the Invention The present invention relates to the finding that arylimidothiazole derivative inhibitors of alkaline phosphatase can reduce neuronal death induced by ,~-amyloid peptide.
In a first aspect, the present invention is a method of inhibiting ,6-amyloid toxicity in brain cells. This method includes administering to the cells an amount of an arylimidothiazole derivative having alkaline phosphatase inhibitory activity effective to reduce cell degeneration. The brain cells can be located in a living organism suffering from an amyloidogenic neurologic disorder, such as Alzheimer's Disease (AD). The brain cells can also be located in a living organism suffering from other amyloidogenic diseases, such as Downs Syndrome or Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch Type (HCHWA-D). The brain cells can also be located in vitro. In a preferred embodiment, the arylimidothiazole derivative is administered orally, intravenously, intraperitoneally, intramuscularly, by injection into cerebrospinal fluid, by injection into an intracerebral ventricle, or via a carrier protein. The arylimidothiazole derivative is preferably administered at a dose of from approximately 0.01 mg/kg to 10 mg/kg body weight per day, more preferably less than 5 mg/kg. Preferred arylimidothiazole derivatives include levamisole, bromolevamisole, and bromotetramisole.
In a second aspect, the invention is a method of treating a human subject havinga peripheral neuropathy. This method includes administering to the subject an amount ~ ~ r~- -2 ~ 6 3 9 6 6 PCT/US94/06186 of an alkaline phosphatase inhibitor which is pharmacologically effective to alleviate the symptoms of the peripheral neuropathy. The alkaline phosphatase inhibitor can beadministered in any of a variety of ways, including orally, intravenously, intraperitoneally, or intramuscularly. Preferred alkaline phosphatase inhibitors for this aspect of the 5 invention are arylimidothiazole derivatives, such as levamisole, bromolevamisole and bromotetramisole. The alkaline phosphatase inhibitor is preferably administered at a dose of from approximately 0.001 mg/kg to 10 mg/kg body weight per day, more preferably less than 5 mg/kg.
In a third aspect the invention is a method of treating a human subject having aperipheral neuropathy. This method includes administering to the subject an amount of an acid phosphatase inhibitor which is pharmacologically effective to alleviate the symptoms of the peripheral neuropathy. The acid phosphatase inhibitor can be administered in any of a variety of ways, including orally, intravenously, intraperitoneally, or intramuscularly. Preferred acid phosphatase inhibitors for this aspect of the invention 1 5 are para-chloromercuribenzoate (pCMB) and inorganic salts of tal l, ale or fluoride.
Preferred inorganic salts for this aspect of the invention are Na; K; and Na,K. The acid phosphatase inhibitor is preferably administered at a dose of from approximately 0.001 mg/kg to 10 mg/kg body weight per day.
Brief DescriPtion of the Drawinqs FIGURE 1 graphically depicts the effect of alkaline phosphatase inhibitors on rat brain neurotumor cells treated with ~-amyloid peptide.
FIGURE 2 graphically depicts the effect of levamisole on primary cultures of ratembryonic hippocampal neurons exposed to ~-amyloid peptide.
FIGURE 3 graphically depicts the effect of okadaic acid on hippocampal neurons treated with,~-amyloid peptide.
FIGURE 4 graphically depicts the effect of levamisole or vanadate on alkaline phosphatase activity from porcine kidney and from calf intestine.
FIGURE 5 graphically depicts the effect of Na,K-ta~l~ate on dorsal root ganglioncells treated with Nerve Growth Factor and vinblastine.
Detailed Description of the Invention The present invention relates to the use of alkaline phosphatase inhibitors or acid phosphatase inhibitors as therapeutic agents for neurological disorders, such as Alzheimer's Disease (AD) and peripheral neuropathies.
It is one of the surprising discoveries of the present invention that arylimidothiazole derivatives having alkaline phosphatase inhibitory activity can be used to inhibit the death of neurons due to,~-amyloid peptide toxicity.
~ 639 66;
WO 94/27603 . = . PCTIUS94/06186 A number of different approaches can be taken to the inhibition of alkaline phosphatases. For example, they can be inhibited by arylimidothiazole compounds such as levamisole, which is specific for mammalian alkaline phosphatase, or by compounds that complex active Zn ion, which is required for alkaline phosphatase activity. Chemical 5 inhibitors include, but are not limited to,~ ~vàmisole I (-)-(S)-2,3,5,6-tetrahydro-6-phenylimidoazide [2,1-b] thiazole -rnonohydrochloride), bromolevamisole, bromotet,a",isole, other arylimidothiazole derivatives, and the like. Still other alkaline phosphatase inhibitors include 1,10 phenanthroline, phenylalanine, IBMX, purines, and theophylline.
The presence of ,6-amyloid in AD and other amyloidogenic proteins is associated not only with deposition of amyloid in plaques, but also with neurotoxicity associated with this peptide. Thus, prevention of this neurotoxicity is an important goal of the present invention. We have surprisingly discovered that arylimidothiazole derivatives having alkaline phosphatase inhibitory activity can attenuate or prevent such 1 5 neurotoxicity.
Another aspect of the present invention involves administration of acid phosphatase inhibitors to patients with peripheral neuropathies.
A number of different approaches can be taken to the inhibition of acid phosphatases. Acid phosphatase can be inhibited by chemical compounds, such as para-chloromercuribenzoate (pCMB) or inorganic salts of tartrate and fluoride. Preferred inorganic salts include Na; K; and Na,K.
One aspect of the present invention involves administration of inhibitors of alkaline phosphatase to patients with an amyloidogenic disease associated with ,B-protein, including AD, Downs Syndrome and HCHWA-D, as well as administration to patients suspected of having these diseases, particularly AD.
Alzheimer's Disease can be diagnosed by any number of traditional diagnostic criteria such as psychometrics that assess, for example, cognitive function, intelligence, language, memory, visual-spatial skills, and frontal lobe skills, or by neuropathological criteria such as immunological markers, enzyme assay, or CAT scan.
The invention can also be used prophylactically for those patients at risk for the development of AD or other amyloidogenic disease. Such patients can be identified through family histories or through a biochemical or immunological test indicative of such diseases. An example of an immunological test for AD involves the use of an antibody for the amyloid precursor protein (APP) in the CSF of a patient suspected of having AD.
In this test, low levels of APP compared to normal levels are indicative of AD. See Van Nostrand et al., Science, 256:1279 (1992).
WO 94/27603 2 1 6 3 9 6 ~ ~` PCT/US94/06186 The administration of alkaline phosphatase inhibitors to prevent~-amyloid peptide toxicity can be by any of a number of methods known to those skilled in the art. Such methods include the delivery of the inhibitor orally or through intravenous, intraperitoneal, or intramuscular injection. Other known methods include techniques for administering compounds directly into the central nervous system (CNS), such as injection intocerebrospinal fluid, injection into an intracerebral ventricle, or by a carrier protein that actively transports the inhibitor to the brain. Admihistration of alkaline or acid phosphatase inhibitors for treatment of peripheral neuropathies can be by any of the foregoing; however, such treatment generally does not involve admir~ ion into the 1 0 CNS.
P~efer,ed dosage range when using chemical inhibitors can be determined using techniques known to those having ordinary skill in the art. For levamisole, dosages of 0.001 mg/kg to 10 mg/kg body weight per day for an average adult, more preferably less than 5 mg/kg, are believed to be effective in the treatment of AD. For some patients daily doses of an alkaline phosphatase inhibitor is required for optimum relief. For other patients relief can be achieved with every other day, or even weekly administration.
It is known that cultured rat hippocampal neurons are killed by the application of ~-amyloid peptides. Thus, prevention of neurotoxicity can be shown as an inhibition in death of these neurons. Selection of appropriate alkaline phosphatase inhibitors for use in inhibitors of ,~-amyloid toxicity can thus be made through use of a model system making use of hippocampal neurons in the presence of,~-amyloid peptides. Compounds that prevent death of these neurons in this system are likely to have significant activity in inhibition of ~-amyloid toxicity.
The effect of treatment with alkaline phosphatase inhibitors on neurons exposed to,t~-amyloid peptide was tested in two experiments, described below in Examples 1 and 2.
Examnle 1 Inhibition of ~-amYloid PePtide Toxicitv bY Alkaline Phosphatase Inhibitors HT4 neurotumor cells were treated with 50~uM,B-amyloid peptide 25-35 and 250 ~M quisqualate. The cells were also treated with 100 ~M of the following alkaline phosphatase inhibitors: 1,10 phenanthroline, phenylalanine, IBMX, bromotetramisole, purine, and theophylline. The death of the neurons was determined by measuring LDH
activity in the media 24 hours after treatment. The release of LDH from the cells into the surrounding media indicates cell death.
WO 94/27603 2 ~ 6 3 9 6 6 s PCTIUS94/06186 FIGURE 1 illustrates the results of the study of Example 1. Control cells, treated only with,~-amyloid peptide and quisqualate, showed a high LDH activity 24 hours after treatment, indicating a high degree of cell death. Cells subjected to no treatment had only a low level of LDH activity. The addition of alkaline phosphatase inhibitors 5 significantly reduced the amount of LDH act~vity and therefore inhibited the death of neuronal cells due to exposure to ,B-amyloid peptide. IBMX appeared to be the most effective compound tested in inhibiting cell death from amyloid toxicity.
ExamPle 2 Inhibition of B-amvloid PePtide Toxicitv bv Levamisole To further determine the effect of alkaline phosphatase inhibitors on ~-amyloid peptide toxicity, rat embryonic hippocampal neurons in culture were exposed to 50,uM
,B-amyloid 25-35 in the presence of levamisole concentrations of 250 ~M and 500,uM.
The results of the experiment of Example 2 are illustrated in FIGURE 2. The death 15of the neurons, as indicated by the release of LDH from the cells, was reduced by the presence of levamisole at a concentration of 250,uM. LDH activity was also reduced by the presence of levamisole at a concentration of 500,uM.
Example 2 demonstrates that death of neuronal cells due to exposure to,B-amyloidpeptide is reduced by the presence of levamisole.
20As mentioned earlier, the prior art indicates that okadaic acid may be useful in the treatment of amyloidosis associated with Alzheimer's Disease. The experiment of Example 3 below, however, illustrates that okadaic acid causes hippocampal neuronal injury upon administration with ,B-amyloid.
25ExamPle 3 Okadaic Acid Causes HiPPocamPal Neuronal Iniurv UPon Administration with ~-Amvloid Hippocampal neurons were treated with 50,uM ,B-amyloid (,~25-35) and 50 nM
okadaic acid for 24 hours in Dulbeccois Modified Eagle's (DME) medium at 37C.
30Following the 24-hour treatment period, an aliquot of media was removed for determination of LDH efflux from the neurons, which is an indicator of cell death. Each bar in FIGURE 3, except the bar representing okadaic acid alone, represents the mean of 4 wells ~t the standard deviation. The bar representing okadaic acid alone represents the mean of 2 wells ~ the standard deviation.
35FIGURE 3 illustrates that okadaic acid causes hippocampal neuronal injury uponadministration with ~-amyloid. This finding is particularly surprising in light of the prior WO 94/27603 ~ t 6 3 9 6 6 = PCT/US94/06186 art, wherein okadaic acid is indicated as being useful for the treatment of amyloidosis associated with Alzheimer's Disease.
The effect of different alkaline phosphatase inhibitors are compared below in Example 4.
Example 4 Inhibition of Porcine Kidnev and Calf Intestinal Alkaline PhosPhatase Porcine kidney (0.01 U) and calf intestinal (0.002 U) alkaline phosphatases wereassayed in the presence of levamisole (200,uM) or vanadate salt (5-500,uM) using pNPP
(5 mM) as sub~l~ate. The assay was carried out in pH 5.5 Iysis buffer. Each bar in FIGURE 4 represents the mean of quadruplicates i the standard deviation.
The results of the experiment of Example 4 are illustrated in FIGURE 4. Both levamisole and vanadate appear to inhibit porcine kidney alkaline phosphatase activity 15 from porcine kidney and calf intestine. Inhibition of porcine kidney alkaline phosphatase activity is unexpectedly high in the presence of levamisole. Vanadate at 100,uM
significantly inhibited calf intestinal alkaline phosphatase, while levamisole at 200~M had little effect.
The effec~ of treatment on humans with AD using arylimidothiazole derivatives 20 having alkaline phosphatase inhibitory activity is described below in Examples 5-9.
Example 5 Oral Treatment of AD with Arvlimidothiazole Derivatives Human subjects diagnosed as suffering from AD are administered 50 mg of an 25 arylimidothiazole derivative, such as levamisole, orally every eight hours for three days.
The treatment is repeated every 14 days for a period of 6 months. With treatment, the symptoms of AD in the subject are lessened, as the subject experiences improved mernory and cognitive skills.
ExamPle 6 Intravenous Treatment of AD
with ArYlimidothiazole Derivatives Human subjects diagnosed as suffering from AD are administered 0.5 mg/kg arylimidothiazole derivatives intravenously once or twice daily for four days. This administration is repeated every 14 days for a period of 4 months.
- . ~
WO 94/27603 2 1 6 3 9 ~ ~ -8- PCT/US94/06186 Example 7 Treatment of AD with Levamisole A human patient diagnosed as suffering from Alzheimer's Disease is administered about 50 mg levamisole orally every eight hours for three days. The treatment is5 repeated every 14 days for a period of 6 months. The symptoms of AD in the patient are lessened, as the patient experiences improved memory and cognitive skills.
ExamPle 8 Continuous Treatment of AD
with Arylimidothiazole Derivatives Human subjects diagnosed as suffering from AD are administered 0.05 mg/kg of an arylimidothiazole derivative, such as levamisole, orally every day for a period of six months. With treatment, the symptoms of AD in the patient decrease and deterioration stops shortly after initiation of treatment.
ExamPle 9 Continuous Treatment of AD with Levamisole A human patient diagnosed as suffering from Alzheimer's Disease is identified ashaving deteriorating symptoms of the disease. The patient is administered about 10 mg 20 levamisole orally once per day for a period of six months. With treatment, the symptoms of AD in the patient decrease and deterioration stops shortly after initiation of treatment.
As discussed above, the present invention includes the treatment of peripheral neuropathies, such as chemotherapeutic induced neuropathy, and neurophathies caused by diabetes and other degenerative disease states. In the context of this aspect of the 25 invention, both acid and alkaline phosphatase inhibitors are effective. Exemplary inhibitors in this regard include Na, K-tartrate, NaF, calyculin-A, vanadate and the arylimidothiazole derivatives. Identification of appropriate protein phosphatase inhibitors can be conducted through a model system making use of dorsal root ganglion cellstreated with Nerve Growth Factor (NGF) and vinblastine. Effective compounds in this 30 system result in an increased percentage of cell viability in the presence of vinblastine.
The effect of treatment with Na,K-tartrate on dorsal root ganglion cells treatedwith Nerve Growth Factor and vinblastine was tested and is described below in Example 10.
WO 94/27603 2 t 6 3 9 6 6 ~ . ~ PCT/TJSg4/06186 g ExamPle 10 Inhibition of Vinblastine-lnduced Toxicity bv Na,K-Tartrate Dorsal root ganglion cells were treated with Nerve Growth Factor (NGF) (50 ng/ml), vinblastine (500 nM) and Na,K-tartrate (500 ,L~M) for 24 hours. Following the treal ment, the dorsal root ganglion cells were stained with ethidium bromide and fluorescein diacetate to stain dead and live cells, respectively.
FIGURE 5 illustrates the results of the study of Example 16. Percentage cell viability is determined from the ratio of live cells to total cell number. Each bar in FIGURE
7 represents the mean of 3 wells i the standard deviation. Controlcells,treatedonly with NGF, showed a high degree of cell viability. Cells exposed to NGF and vinblastine showed a decrease in cell viability. The addition of Na,K-tartrate significantly increased the percentage cell viability in the presence of vinblastine.
The effect of treatment on humans with peripheral neuropathy using either alkaline or acid phosphatase inhibitors is described below in Examples 1 1-14.
Example 1 1 Treatment of ChemotheraPeutic-lnduced PeriPheral Neuropathv with Acid Phosphatase Inhibitors Human subjects with peripheral neuropathy caused by chemotherapeutic agents such as vinblastine, vincristine, or taxol are administered 10 mg/kg of an acid phosphatase inhibitor. The acid phosphatase inhibitor is administered orally or systemically to these subjects once or twice a day for two days prior to and four days following the administration of the chemotherapeutic regimen. Following treatment, an assessment of motor coordination and peripheral paresthesia is made, as specified by those skilled in the art.
ExamPle 12 Treatment of NonchemotheraPeutic-lnduced PeriPheral NeuroPathv with Acid Phosphatase Inhibitors Human subjects with peripheral neuropathy caused by diabetes or other degenerative disease states are administered 5 mg/kg of an acid phosphatase inhibitor twice per day for a period of 6 months. Following treatment, an assessment of motor coordlination and peripheral paresthesia is made, as specified by those skilled in the art.
W094/27603 ~l 63966 PCT/US94/06186 ExamDIe 13 Treatment of ChemotheraPeutic-lnduced PeriPheral NeuroPathY with Alkaline Phosphatase Inhibitors Human subjects with peripheral neuropathy caused by chemotherapeutic agents 5 such as vinblastine, vincristine, or taxol are administered 5 mg/kg of levamisole. The alkaline phosphatase inhibitor is administered orally or systemically to these subjects once or twice a day for two days prior to and'~four days following the administration of the chemotherapeutic regimen. Following trèatment, an assessment of motor coordination and peripheral paresthesia is made, as specified by those skilled in the art.
Example 14 Treatment of Nonchemotherapeutic-lnduced PeriPheral NeuroPathY with Alkaline Phosphatase Inhibitors Human subjects with peripheral neuropathy caused by diabetes or other 15 degenerative disease states are administered 5 mg/kg of an alkaline phosphatase inhibitor. The alkaline phosphatase inhibitor is administered orally or systemically to the subjects once or twice a day for two days prior to and four days following the administration of the chemotherapeutic regimen. Following treatment, an assessment of motor coordination and peripheral paresthesia is made, as specified by those skilled in the 20 art.
Claims (30)
1 . Use of an arylimidothiazole derivative having alkaline phosphatase inhibitory activity in the preparation of a medicament for inhibiting .beta.-amyloid toxicity in brain cells exposed to .beta.-amyloid peptide.
2. The use of Claim 1, wherein said brain cells are located in a living organismsuffering from a neurologic disorder.
3. The use of Claim 2, wherein said neurologic disorder is Alzheimer's Disease.
4. The use of Claim 1, wherein said brain cells are located in a living organismsuffering from an amyloidogenic disease associated with .beta.-protein.
5. The use of Claim 4, wherein said amyloidogenic disease is Downs Syndrome or Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch Type.
6. The use of Claim 1, wherein said brain cells are located in vitro.
7. The use of Claim 2, wherein the medicament is for administration orally, intravenously, intraperitoneally, intramuscularly, by injection into cerebrospinal fluid, by injection into an intracerebral ventricle, or via a carrier protein.
8. The use of Claim 1, wherein said arylimidothiazole derivative is levamisole, bromolevamisole or bromotetramisole.
9. Use of an alkaline or acid phosphatase inhibitor in the preparation of a medicament for treating a human subject having a peripheral neuropathy.
10. The use of Claim 9, wherein the medicament is for administration orally, intravenously, intraperitoneally, intramuscularly, by injection into cerebrospinal fluid, by injection into an intracerebral ventricle, or via a carrier protein.
11. The use of Claim 9, wherein said phosphatase inhibitor is an arylimidothiazole derivative.
12. The use of Claim 11, wherein said arylimidothiazole derivative is levamisole, bromolevamisole or bromotetramisole.
13. The use of Claim 9, wherein said acid phosphatase inhibitor is para chloro mercuribenzoate (PCMB) or an inorganic salt of tartrate or fluoride.
14. The use of Claim 13, wherein said inorganic salt is of Na; K; or Na,K.
15. A method of inhibiting .beta.-amyloid toxicity in brain cells exposed to .beta.-amyloid peptide, comprising:
administering to said cells an arylimidothiazole derivative having alkaline phosphatase inhibitory activity in an amount effective to reduce cell degeneration.
administering to said cells an arylimidothiazole derivative having alkaline phosphatase inhibitory activity in an amount effective to reduce cell degeneration.
16. The method of Claim 15, wherein said brain cells are located in a living organism suffering from a neurologic disorder.
17. The method of Claim 16, wherein said neurologic disorder is Alzheimer's Disease.
18. The method of Claim 15, wherein said brain cells are located in a living organism suffering from an amyloidogenic disease associated with .beta.-protein.
19. The method of Claim 18, wherein said amyloidogenic disease is Downs Syndrome or Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch Type.
20. The method of Claim 15, wherein said brain cells are located in vitro.
21. The method of Claim 16, wherein the administering step comprises administering said arylimidothiazole derivative orally, intravenously, intraperitoneally, intramuscularly, by injection into cerebrospinal fluid, by injection into an intracerebral ventricle, or via a carrier protein.
22. The method of Claim 16, wherein said arylimidothiazole derivative is administered at a dose of from approximately 0.01 mg/kg to 5 mg/kg body weight per day.
23. The method of Claim 15, wherein said arylimidothiazole derivative is levamisole, bromolevamisole or bromotetramisole.
24. A method of treating a human subject having a peripheral neuropathy, comprising:
administering to said subject an amount of an alkaline or acid phosphatase inhibitor which is pharmacologically effective to alleviate the symptoms of the peripheral neuropathy.
administering to said subject an amount of an alkaline or acid phosphatase inhibitor which is pharmacologically effective to alleviate the symptoms of the peripheral neuropathy.
25. The method of Claim 24, wherein the administering step comprises administering said alkaline phosphatase inhibitor orally, intravenously, intraperitoneally, intramuscularly, by injection into cerebrospinal fluid, by injection into an intracereberal ventricle, or via a carrier protein.
26. The method of Claim 24, wherein said alkaline phosphatase inhibitor is administered at a dose of from approximately 0.001 mg/kg to 10 mg/kg body weight per day.
27. The method of Claim 24, wherein said alkaline phosphatase inhibitor is an arylimidothiazole derivative.
28. The method of Claim 27, wherein said arylimidothiazole derivative is selected from the group consisting of levamisole, bromolevamisole and bromotetramisole.
29. The method of Claim 24, wherein said acid phosphatase inhibitor is an inorganic salt of tartrate or fluoride.
30. The method of Claim 29, wherein said inorganic salt is selected from the group consisting of Na; K; and Na,K.
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| US4939124A (en) * | 1988-01-26 | 1990-07-03 | Laboratorios Serono S.A. | Treatment and diagnosis of dementia |
| JP2675573B2 (en) * | 1988-03-31 | 1997-11-12 | 科研製薬株式会社 | Brain function improver |
| US4837164A (en) * | 1988-04-27 | 1989-06-06 | Bionix Corporation | Methods for diagonosing, monitoring and controlling the onset and progression of certain dementias and impeding memory loss or improving impairment of memory |
| US5151508A (en) * | 1988-08-30 | 1992-09-29 | Molecular Therapetuics, Inc. | Promoter region for DNA sequences which encode the precursor of the human A4 amyloid protein |
| US4973591A (en) * | 1988-10-21 | 1990-11-27 | Syntex Pharmaceuticals, Ltd. | Parenteral formulations of 1-diphenylmethyl-4-((2-(4-methylphenyl)-5-methyl-1H-imidazol-4-yl)methyl)piperazine |
| US5063220A (en) * | 1988-10-21 | 1991-11-05 | Syntex Pharmaceuticals, Ltd. | Parenteral formulations of 1-diphenylmethyl-4-((2-(4-methylphenyl)-5-methyl-1H-imidazol-4-yl)methyl)piperazine |
| US5135847A (en) * | 1988-11-18 | 1992-08-04 | Becton, Dickinson And Company | Substrate composition for alkaline phosphatase and method for assay using same |
| US5100654A (en) * | 1989-04-07 | 1992-03-31 | Yale University | Phosphorylated derivatives of l-dopa and compositions and methods for increasing the melanin content in mammalian skin and hair |
| US5234814A (en) * | 1989-06-01 | 1993-08-10 | Du Pont Merck Pharmaceutical Company | Diagnostic assay for alzheimer's disease |
| US5260210A (en) * | 1989-09-27 | 1993-11-09 | Rubin Lee L | Blood-brain barrier model |
| EP0527823A1 (en) * | 1990-04-24 | 1993-02-24 | The Regents Of The University Of California | Purification, detection and methods of use of protease nexin-2 |
| JPH0725786A (en) * | 1990-05-16 | 1995-01-27 | Univ Rockefeller | Medical treatment of amyloidosis accompanying alzheimer desease |
| US5200324A (en) * | 1990-09-04 | 1993-04-06 | E. R. Squibb & Sons, Inc. | Method of diagnosing senile dementia of the Alzheimer type |
| US5246944A (en) * | 1991-08-13 | 1993-09-21 | Merck & Co., Inc. | Quinoline angiotensin ii antagonists incorporating a substituted benzyl element |
| US5206260A (en) * | 1991-11-07 | 1993-04-27 | Merck & Co., Inc. | Hexahydropyrrolo[2,3-b]indole derivatives |
-
1994
- 1994-06-01 JP JP7501063A patent/JPH08511005A/en active Pending
- 1994-06-01 EP EP94919323A patent/EP0701441A1/en not_active Withdrawn
- 1994-06-01 CA CA002163966A patent/CA2163966A1/en not_active Abandoned
- 1994-06-01 WO PCT/US1994/006186 patent/WO1994027603A1/en not_active Application Discontinuation
- 1994-06-01 US US08/252,109 patent/US5567724A/en not_active Expired - Lifetime
- 1994-06-01 AU AU70506/94A patent/AU698101B2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| JPH08511005A (en) | 1996-11-19 |
| AU7050694A (en) | 1994-12-20 |
| EP0701441A1 (en) | 1996-03-20 |
| AU698101B2 (en) | 1998-10-22 |
| WO1994027603A1 (en) | 1994-12-08 |
| US5567724A (en) | 1996-10-22 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |